During mitosis in eukaryotic cells, sister chromatids segregate to opposite poles. Failure to properly segregate all the chromosomes leads to aneuploidy which can result in cell death, birth defects or contribute to tumor formation. One of the least understood, but essential aspects of mitotic chromosome segregation is the mechanism used to hold sister chromatids together until their segregation is triggered at anaphase. In this proposal, we will utilize Saccharomyces cerevisiae to isolate temperature sensitive mutants defective in sister chromatid cohesion. Two primary screens will be utilized. First, we will isolate mutants which exhibit enhanced lethality following transient mitotic arrest but not following transient GI arrest. Mutants undergoing precocious chromatid segregation will result in cell death specifically at mitosis and so should be identified by the screen. Second, we will use fluorescence microscopy to identify mutants exhibiting unequal or premature separation of the bulk of chromosomal DNA. Mutants identified by either screen will be subjected to additional cytological, genetic and biochemical assays to verify their role in sister chromatid association. The genes that complement the sister chromatid cohesion mutants will be cloned and used to characterize the molecular and biochemical properties of their products. The gene products from these clones will be purified by expressing them in E. coli and antibodies to these products made. Using these tools it will be possible to ask basic questions about the nature of the molecular glue used to hold sister chromatids together and the mechanism of its regulation.